Dairy Chemistry and Biochemistry

P.F. Fox T. Uniacke-Lowe P.L.H. McSweeney J.A. O’Mahony Dairy Chemistry and Biochemistry Second Edition

Dairy Chemistry and Biochemistry



P.F. Fox • T. Uniacke-Lowe P.L.H. McSweeney • J.A. O’Mahony Dairy Chemistry and Biochemistry Second Edition

P.F. Fox T. Uniacke-Lowe School of Food and Nutritional Sciences School of Food and Nutritional Sciences University College University College Cork, Ireland Cork, Ireland P.L.H. McSweeney J.A. O’Mahony School of Food and Nutritional Sciences School of Food and Nutritional Sciences University College University College Cork, Ireland Cork, Ireland ISBN 978-3-319-14891-5 ISBN 978-3-319-14892-2 (eBook) DOI 10.1007/978-3-319-14892-2 Library of Congress Control Number: 2015933835 Springer Cham Heidelberg New York Dordrecht London © Springer International Publishing Switzerland 2015 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. Printed on acid-free paper Springer International Publishing AG Switzerland is part of Springer Science+Business Media (www.springer.com)

Preface to First Edition Milk has been the subject of scientific study for about 150 years and, consequently, is probably the best characterized, in chemical terms, of our major foods. It is prob- ably also the most complicated and serves as the raw material for a very large and diverse family of food products. Dairy science has existed as a university discipline for more than 100 years; it is the oldest sector of food science (and technology), with the exception of brewery science. Since dairy chemistry is a major facet of dairy science, it might be expected to have been the subject of numerous books. This is, in fact, not so. During the past 40 years, as far as we are aware, only six books or series on dairy chemistry have been published in English, i.e. Principles of Dairy Chemistry (Jenness and Patton 1959), Dairy Chemistry and Physics (Walstra and Jenness 1984), Fundamentals of Dairy Chemistry (Webb and Johnson 1964; Webb et al. 1974; Wong et al. 1988), Developments in Dairy Chemistry (Fox, four volumes, 1982, 1983, 1985, 1989), Advanced Dairy Chemistry (Fox, three volumes, 1992, 1995, 1997) and Handbook of Milk Composition (Jensen 1995). Of these, Principles of Dairy Chemistry and Dairy Chemistry and Physics were written essentially for senior undergraduate students. The other four books/series were focussed principally on lecturers, researchers, senior postgraduate students and senior production management. Thus, at present there is a lack of books written at senior undergraduate/junior postgraduate level specializing in dairy chemistry/sci- ence. This book is intended to fill that gap and should be useful to graduates work- ing in the dairy industry as it is to those still studying. This book assumes a knowledge of chemistry and biochemistry but not of dairy chemistry. As the title suggests, the book has a stronger biochemical orientation than either Principles of Dairy Chemistry or Dairy Chemistry and Physics. In addi- tion to a fairly in-depth treatment of the chemistry of the principal constituents of milk, i.e. water, lactose, lipids, proteins (including enzymes), salts and vitamins, various more applied aspects are also covered, e.g. heat-induced changes, cheese, protein-rich products and the applications of enzymes in dairy technology. The prin- cipal physical properties are also described. To facilitate the reader, the structure of various molecules mentioned frequently in the text is given in appendices but we emphasize that a good general knowledge v

vi Preface to First Edition of chemistry and biochemistry is assumed. The chemical composition of the principal dairy products is also included. This book does not cover the technology of various dairy products, although brief manufacturing protocols for some products are included to facilitate discus- sion; however, a number of textbooks on various aspects of dairy technology are referenced. Neither are the chemical analyses, microbiology and nutritional aspects of dairy products covered, except in a very incidental manner. The effects of dairy husbandry on the composition and properties of milk are discussed briefly, as is the biosynthesis of milk constituents; in both cases, some major textbooks are referenced. We hope that the book will answer some of your questions on the chemistry and biochemistry of milk and milk products and encourage you to undertake more extensive study of these topics. The highly skilled and enthusiastic assistance of Ms Anne Cahalane and Ms Brid Considine in the preparation of the manuscript and of Professor D. M. Mulvihill and Dr. Nora O’ Brien for critically and constructively reviewing the manuscript is gratefully acknowledged and very much appreciated. Cork, Ireland P.F. Fox P.L.H. McSweeney

Preface to Second Edition Since the publication of the first edition of this book by Chapman & Hall in 1998, there has been considerable progress on several aspects of the subject. The book was reprinted by Kluwer Academic/Plenum Publishers but not revised and is out of print. All topics covered in the first edition are retained, revised and expanded in the second edition. A new chapter “Bioactive Compounds in Milk” has been added and a full chapter has been devoted to “Fermented Milk Products”, which were part of the chapter “Chemistry and Biochemistry of Cheese and Fermented Milk” in the first edition. The book is focussed on undergraduate and junior post-graduate stu- dents but should also be useful for teaching staff in dairy/food science/technology, researchers and industrial personnel, and for those changing direction. The book assumes a sound knowledge of general and physical chemistry and of biochemistry. The manufacture of the various dairy products mentioned is not described in detail, but the book provides appropriate references on dairy technology. The principles of the main analytical methods for lactose, lipids, proteins and milk salts are presented, but the methods are not described in detail. The nutritional and microbiological aspects of milk and dairy products are discussed only in so far as they are affected by, or affect, the chemistry and biochemistry of milk and dairy products. The effects of dairy husbandry on the composition and properties of milk are discussed briefly, as is the biosynthesis of milk constituents. We expect that the book will answer some of your questions on the chemistry and biochemistry of milk and milk products and stimulate your interest in studying these subjects in greater detail. Cork, Ireland P.F. Fox T. Uniacke-Lowe P.L.H. McSweeney J.A. O’Mahony vii



General References on Dairy Chemistry Alais, C. (1974). Science du Lait. Principes des Techniques Laitieres (3rd ed.). Paris: SEP Editions. Associates of Rogers (1928). Fundamentals of dairy science. American Chemical Society Monograph No. 41. New York: The Chemical Catalog. [1935, New York: Reinhold Publishing; 1955, 2nd ed., New York: Reinhold Publishing]. Cayot, P., & Lorient, D. (1998). Structure et Technofonctions des Proteins du Lait. Paris: Lavoisier Technique and Documentation. Davis, J. G., & MacDonald, F. J. (1955). Richmond’s dairy chemistry (5th ed.). London: Charles Griffin and Company. Fleischmann, W. (1870). Leherbuch der Milchwissenschaft. Bremen: M. Heinsius. [7 editions up to 1932]. Fox, P. F. (Ed.). (1982–1989). Developments in dairy chemistry (Vols. 1–4). London: Elsevier Applied Science Publishers. Fox, P. F. (Ed.). (1992–1997). Advanced dairy chemistry (Vols. 1–3). London: Elsevier Applied Science Publishers and Chapman and Hall. Fox, P. F., & McSweeney, P. L. H. (1998). Dairy chemistry and biochemistry. London: Chapman and Hall. Fox, P. F., & McSweeney, P. L. H. (2003). Advanced dairy chemistry (Vol. 1, Proteins, 3rd ed.). New York: Kluwer Academic/Plenum Publishers Fox, P. F., & McSweeney, P. L. H. (2006). Advanced dairy chemistry (Vol. 1, Lipids, 3rd ed.). New York: Springer. Jenness, R., & Patton, S. (1959). Principles of dairy chemistry. New York: John Wiley & Sons. Jensen, R. G. (ed.) (1995). Handbook of milk composition. San Diego: Academic Press. Ling, E. R. (1930). A textbook of dairy chemistry. London: Chapman and Hall. (3 further reprints/editions, 1944, 1949, 1956). McKenzie, H. A. (1970, 1971). Milk proteins: Chemistry and molecular biology (Vols. 1 and 2). New York: Academic Press. McSweeney, P. L. H., & Fox, P. F. (2009). Advanced dairy chemistry (Vol. 3, Lactose, water, salts and minor constituents, 3rd ed.). New York: Springer ix

x General References on Dairy Chemistry McSweeney, P. L. H., & Fox, P. F. (2013). Advanced dairy chemistry (Vol. 1A, Proteins, basic aspects, 4th ed.). New York: Springer. McSweeney, P. L. H., & O’Mahoney, J. A. (Eds.). (2015). Advanced dairy chemis- try (Vol. 1, Proteins, Part B). New York: Springer. Richmond, H. D. (1899). Dairy chemistry: A practical handbook for dairy chemists and others having control of dairies. London: C Griffin and Company. (pub- lished in 5 editions, the 5th, revised by Davis and MacDonald in 1955, see above). Singh, H., Boland, B., & Thompson, A. (2014). Milk proteins: From expression to food (2nd ed.). San Diego: Academic Press. Snyder, H. (1897). The chemistry of dairying. Easton, PA: Chemical Publishing Company. Thompson, A., Boland, B., & Singh, H. (2009). Milk proteins: From expression to food. San Diego: Academic Press. Walstra, P., Geurts, T. J., Noomen, A., Jellema, A., & van Boeckel, M. A. J. S. (1999). Dairy technology: Principles of milk properties and processes. Marcel Dekker, New York. Walstra, P., & Jenness, R. (1984). Dairy chemistry and physics. New York: John Wiley & Sons. Walstra, P., Wouters, J. T. H., & Geurts, T. J. (2005). Dairy science and technology. Oxford: CRC/Taylor and Francis. Webb, B. H., & Johnson, A. H. (Eds.). (1964). Fundamentals of dairy chemistry. Westport, CT, USA: AVI. Webb, B. H., Johnson, A. H., & Alford, J. A. (Eds.). (1974). Fundamentals of dairy chemistry (2nd ed.). Westport, CT, USA: AVI. Wong, N. P., Jenness, R., Keeney, M., & Marth, E. H. (Eds.). (1988). Fundamentals of dairy chemistry (3rd ed.). New York: Van Nostrand Reinhold.

Contents 1 Production and Utilization of Milk ....................................................... 1 1.1 Introduction...................................................................................... 1 1.2 Composition and Variability of Milk ............................................... 1 1.3 Classification of Mammals............................................................... 3 1.4 Structure and Development of Mammary Tissue ......................................................................... 4 1.5 Ultrastructure of the Secretory Cell ................................................. 7 1.6 Techniques Used to Study Milk Synthesis....................................... 8 1.6.1 Arterio-Venous Concentration Differences.......................... 8 1.6.2 Isotope Studies ..................................................................... 9 1.6.3 Perfusion of Isolated Gland.................................................. 9 1.6.4 Tissue Slices......................................................................... 9 1.6.5 Cell Homogenates ................................................................ 9 1.6.6 Tissue Culture ...................................................................... 10 1.7 Biosynthesis of Milk Constituents ................................................... 11 1.8 Production and Utilization of Milk .................................................. 11 1.9 Trade in Milk Products..................................................................... 15 References................................................................................................. 18 Suggested Reading.................................................................................... 19 2 Lactose ..................................................................................................... 21 2.1 Introduction...................................................................................... 21 2.2 Chemical and Physical Properties of Lactose......................................................................................... 24 2.2.1 Structure of Lactose ............................................................. 24 2.2.2 Biosynthesis of Lactose........................................................ 26 2.2.3 Lactose Equilibrium in Solution .......................................... 27 2.2.4 Significance of Mutarotation................................................ 28 2.2.5 Solubility of Lactose ............................................................ 28 2.2.6 Crystallization of Lactose .................................................... 29 2.2.7 Problems related to Lactose Crystallization......................... 32 xi

xii Contents 2.3 Production of Lactose..................................................................... 40 2.4 Derivatives of Lactose .................................................................... 43 43 2.4.1 Enzymatic Modification of Lactose.................................. 44 2.4.2 Chemical Modifications.................................................... 48 2.4.3 Fermentation Products ...................................................... 53 2.5 Lactose and the Maillard Reaction ................................................. 56 2.6 Nutritional Aspects of Lactose ....................................................... 56 2.6.1 Lactose Intolerance........................................................... 59 2.6.2 Galactosemia..................................................................... 60 2.7 Determination of Lactose Concentration........................................ 61 2.7.1 Polarimetry ....................................................................... 61 2.7.2 Oxidation and Reduction Titration ................................... 63 2.7.3 Infrared (IR) Spectroscopy ............................................... 63 2.7.4 Colorimetric Methods....................................................... 63 2.7.5 Chromatographic Methods ............................................... 63 2.7.6 Enzymatic Methods .......................................................... 64 2.8 Oligosaccharides............................................................................. 66 References ................................................................................................ 68 Suggested Reading ................................................................................... 3 Milk Lipids .............................................................................................. 69 3.1 Introduction .................................................................................... 69 3.2 Factors that Affect the Fat Content of Bovine Milk ....................... 70 3.3 Classes of Lipids in Milk................................................................ 72 3.4 Fatty Acid Profile of Milk Lipids ................................................... 76 3.5 Synthesis of Fatty Acids in Milk Fat .............................................. 82 3.6 Structure of Milk Lipids ................................................................. 86 3.7 Milk Fat as an Emulsion................................................................. 90 3.8 Milk Fat Globule Membrane .......................................................... 91 3.8.1 Isolation of the Fat Globule Membrane............................ 92 3.8.2 Gross Chemical Composition of FGM ............................. 93 3.8.3 The Protein Fraction ......................................................... 93 3.8.4 The Lipid Fraction ............................................................ 95 3.8.5 Other Membrane Components.......................................... 96 3.8.6 Membrane Structure ......................................................... 97 3.8.7 Secretion of Milk Lipid Globules ..................................... 99 3.9 Stability of the Milk Fat Emulsion ................................................. 102 3.9.1 Emulsion Stability in General........................................... 102 3.9.2 The Creaming Process in Milk ......................................... 104 3.10 Influence of Processing Operations on the Fat Globule Membrane ......................................................................... 107 3.10.1 Milk Supply: Hydrolytic Rancidity .................................. 107 3.10.2 Mechanical Separation of Milk ........................................ 109 3.10.3 Homogenization................................................................ 112 3.10.4 Heating.............................................................................. 115

Contents xiii 3.11 Physical Defects in Milk and Cream .............................................. 115 3.11.1 Free Fat ............................................................................. 116 3.12 Churning ......................................................................................... 117 3.12.1 Buttermilk ......................................................................... 121 3.13 Freezing .......................................................................................... 122 3.14 Dehydration .................................................................................... 122 3.15 Lipid Oxidation .............................................................................. 123 3.15.1 Autocatalytic Mechanism ................................................. 123 3.15.2 Pro-oxidants in Milk and Milk Products .......................... 126 3.15.3 Antioxidants in Milk......................................................... 128 3.15.4 Spontaneous Oxidation..................................................... 128 3.15.5 Other Factors that Affect Lipid Oxidation in Milk and Dairy Products ........................................................... 129 3.15.6 Measurement of Lipid Oxidation...................................... 130 3.16 Rheology of Milk Fat ..................................................................... 130 3.16.1 Fatty Acid Profile and Distribution................................... 130 3.16.2 Process Parameters ........................................................... 132 3.17 Analytical Methods for the Quantitative Determination of Milk Fat ...................................................................................... 135 3.18 Appendix A: Principal Fatty Acids in Milk Fat.............................. 138 3.19 Appendix B..................................................................................... 139 3.20 Appendix C..................................................................................... 141 References................................................................................................. 141 Suggested Reading.................................................................................... 143 4 Milk Proteins........................................................................................... 145 4.1 Introduction .................................................................................... 145 4.2 Heterogeneity of Milk Proteins ...................................................... 148 4.2.1 Other Protein Fractions..................................................... 149 4.3 Preparation of Casein and Whey Proteins ...................................... 150 4.3.1 Acid (Isoelectric) Precipitation......................................... 150 4.3.2 Centrifugation ................................................................... 151 4.3.3 Centrifugation of Calcium-Supplemented Milk ............... 151 4.3.4 Salting-Out Methods......................................................... 151 4.3.5 Ultrafiltration .................................................................... 152 4.3.6 Microfiltration................................................................... 152 4.3.7 Gel Filtration (Gel Permeation Chromatography)............ 153 4.3.8 Precipitation with Ethanol ................................................ 153 4.3.9 Cryoprecipitation .............................................................. 153 4.3.10 Rennet Coagulation .......................................................... 154 4.3.11 Other Methods for the Preparation of Whey Proteins ...... 154 4.4 Heterogeneity and Fractionation of Casein .................................... 154 4.4.1 Resolution of Caseins by Electrophoresis ........................ 155 4.4.2 Microheterogeneity of the Caseins ................................... 160 4.4.3 Nomenclature of the Caseins ............................................ 162

xiv Contents 4.5 Some Important Properties of the Caseins ..................................... 163 4.5.1 Chemical Composition ..................................................... 163 4.5.2 Secondary and Tertiary Structures.................................... 173 4.5.3 Molecular Size .................................................................. 176 4.5.4 Hydrophobicity ................................................................. 177 4.5.5 Influence of Ca2+ on Caseins............................................. 177 4.5.6 Action of Rennets on Casein ............................................ 178 4.5.7 Casein Association............................................................ 178 4.6 Casein Micelles .............................................................................. 178 4.6.1 Composition and General Features................................... 178 4.6.2 Stability............................................................................. 179 4.6.3 Principal Micelle Characteristics...................................... 181 4.6.4 Micelle Structure .............................................................. 183 4.7 Whey Proteins................................................................................. 187 4.7.1 Preparation........................................................................ 187 4.7.2 Heterogeneity of Whey Proteins....................................... 188 4.8 β-Lactoglobulin .............................................................................. 189 4.8.1 Occurrence and Microheterogeneity................................. 189 4.8.2 Amino Acid Composition ................................................. 189 4.8.3 Primary Structure.............................................................. 190 4.8.4 Secondary Structure.......................................................... 190 4.8.5 Tertiary Structure .............................................................. 190 4.8.6 Quaternary Structure......................................................... 192 4.8.7 Physiological Function ..................................................... 193 4.8.8 Denaturation ..................................................................... 193 4.9 Whey Acidic Protein....................................................................... 193 4.10 α-Lactalbumin ................................................................................ 194 4.10.1 Amino Acid Composition ................................................. 194 4.10.2 Genetic Variants ................................................................ 194 4.10.3 Primary Structure.............................................................. 194 4.10.4 Secondary and Tertiary Structure ..................................... 194 4.10.5 Quaternary Structure......................................................... 196 4.10.6 Other Species .................................................................... 196 4.10.7 Biological Function .......................................................... 196 4.10.8 Metal Binding and Heat Stability ..................................... 196 4.10.9 Apoptosis Effect on Tumour Cells.................................... 196 4.11 Blood Serum Albumin .................................................................... 197 4.12 Immunoglobulins (lg) ..................................................................... 198 4.13 Proteose Peptone 3 ......................................................................... 202 4.14 Minor Milk Proteins ....................................................................... 202 4.15 Non-protein Nitrogen ..................................................................... 202 4.16 Interspecies Comparison of Milk Proteins ..................................... 203 4.17 Synthesis and Secretion of Milk Proteins....................................... 205 4.17.1 Sources of Amino Acids ................................................... 206 4.17.2 Amino Acid Transport into the Mammary Cell................ 206

Contents xv 4.17.3 Synthesis of Milk Proteins................................................ 206 4.17.4 Modifications of the Polypeptide Chain ........................... 209 4.17.5 Structure and Expression of Milk Protein Genes ............. 210 4.17.6 Secretion of Milk-Specific Proteins.................................. 210 4.17.7 Secretion of Immunoglobulins.......................................... 212 4.18 Functional Milk Protein Products................................................... 213 4.18.1 Industrial Production of Caseins....................................... 215 4.18.2 Novel Methods for Casein Production.............................. 219 4.18.3 Fractionation of Casein..................................................... 220 4.18.4 Functional (Physicochemical) Properties of Caseins ....... 221 4.18.5 Applications of Caseins and Whey Proteins..................... 223 4.18.6 Casein-Whey Protein Co-precipitates............................... 223 4.19 Methods for Quantitation of Proteins in Foods .............................. 226 4.19.1 Kjeldahl Method ............................................................... 226 4.19.2 The Formol Titration......................................................... 229 4.19.3 Absorbance of UV Light .................................................. 229 4.19.4 Biuret Method................................................................... 230 4.19.5 Folin-Ciocalteau (F-C) Method ........................................ 230 4.19.6 Dye-Binding Methods ...................................................... 230 4.19.7 Bradford Method .............................................................. 231 4.19.8 Infra-Red Spectroscopy .................................................... 231 4.19.9 Dumas Method.................................................................. 231 Appendix 4A Structures of Amino Acids Occurring in Proteins............. 232 References................................................................................................. 233 Suggested Reading.................................................................................... 238 5 Salts of Milk ............................................................................................ 241 5.1 Introduction .................................................................................... 241 5.2 Method of Analysis......................................................................... 241 5.3 Composition of Milk Salts.............................................................. 242 5.4 Secretion of Milk Salts ................................................................... 244 5.5 Factors Influencing Variation in Salt Composition......................... 245 5.5.1 Breed of Cow .................................................................... 246 5.5.2 Stage of Lactation ............................................................. 246 5.5.3 Infection of the Udder....................................................... 248 5.5.4 Feed................................................................................... 249 5.6 Interrelations of Milk Salt Constituents ......................................... 249 5.6.1 Partition of Milk Salts Between Colloidal and Soluble Phases ........................................................... 251 5.6.2 Methods Used to Separate the Colloidal and Soluble Phases ........................................................... 251 5.6.3 Soluble Salts ..................................................................... 252 5.6.4 Measurement of Calcium and Magnesium Ions ............... 256 5.6.5 Colloidal Milk Salts.......................................................... 257

xvi Contents 5.7 Changes in Milk Salts Equilibrium Induced by Various Treatments...................................................................... 261 5.7.1 Addition of Acid or Alkali.................................................... 261 5.7.2 Addition of Various Salts ..................................................... 262 5.7.3 Effect of Changes in Temperature........................................ 263 5.7.4 Changes in pH Induced by Temperature .............................. 264 5.7.5 Effect of Dilution and Concentration ................................... 265 5.7.6 Effect of Freezing................................................................. 265 5.7.7 Effect of Ultrafiltration......................................................... 266 5.7.8 Effect of High Pressure Processing...................................... 266 5.8 Fortification of Milk with Inorganic Elements................................. 266 5.9 Synthetic Milk Ultrafiltrate .............................................................. 267 References................................................................................................. 267 Suggested Reading.................................................................................... 269 6 Vitamins in Milk and Dairy Products................................................... 271 6.1 Introduction...................................................................................... 271 6.2 Fat-Soluble Vitamins........................................................................ 272 6.2.1 Retinol (Vitamin A).............................................................. 272 6.2.2 Calciferols (Vitamin D)........................................................ 276 6.2.3 Tocopherols and Related Compounds (Vitamin E).............. 278 6.2.4 Phylloquinone and Related Compounds (Vitamin K).......... 280 6.3 B-Group Vitamins ............................................................................ 281 6.3.1 Thiamine (Vitamin B1) ........................................................ 282 6.3.2 Riboflavin (Vitamin B2)....................................................... 284 6.3.3 Niacin ................................................................................... 286 6.3.4 Biotin.................................................................................... 287 6.3.5 Pantothenic Acid .................................................................. 288 6.3.6 Pyridoxine and Related Compounds (Vitamin B6).............. 288 6.3.7 Folate.................................................................................... 291 6.3.8 Cobalamin and Its Derivatives (Vitamin B12) ..................... 294 6.4 Ascorbic Acid (Vitamin C)............................................................... 295 References................................................................................................. 297 7 Water in Milk and Dairy Products........................................................ 299 7.1 Introduction...................................................................................... 299 7.2 General Properties of Water ............................................................. 299 7.3 Water Activity .................................................................................. 306 7.4 Water Sorption ................................................................................. 309 7.5 Glass Transition and the Role of Water in Plasticization ................. 316 7.6 Non-equilibrium Ice Formation ....................................................... 316 7.7 Role of Water in Stickiness and Caking of Powders and Crystallization of Lactose.......................................................... 317 7.8 Water and the Stability of Dairy Products........................................ 317 References................................................................................................. 320 Suggested Reading.................................................................................... 320

Contents xvii 8 Physical Properties of Milk.................................................................... 321 8.1 Ionic Strength................................................................................... 321 8.2 Density ............................................................................................. 322 8.3 Redox Properties of Milk................................................................. 323 8.4 Colligative Properties of Milk.......................................................... 327 8.5 Interfacial Tension............................................................................ 331 8.6 Acid-Base Equilibria........................................................................ 333 8.7 Rheological Properties ..................................................................... 336 8.7.1 Newtonian Behaviour ......................................................... 336 8.7.2 Non-Newtonian Behaviour................................................. 337 8.7.3 Rheology of Milk Gels ....................................................... 338 8.7.4 Rheological Properties of Milk Fat .................................... 339 8.8 Electrical Conductivity ................................................................... 339 8.9 Thermal Properties of Milk ............................................................ 339 8.10 Interaction of Light with Milk and Dairy Products ........................ 340 8.11 Colour of Milk and Milk Products ................................................. 342 References................................................................................................. 343 9 Heat-Induced Changes in Milk ............................................................. 345 9.1 Introduction .................................................................................... 345 9.2 Lipids.............................................................................................. 347 9.2.1 Physico-Chemical Changes ................................................ 347 9.2.2 Chemical Changes .............................................................. 349 9.2.3 Denaturation of Indigenous Enzymes ................................ 350 9.3 Lactose............................................................................................ 350 9.3.1 Formation of Lactulose....................................................... 351 9.3.2 Formation of Acids ............................................................. 351 9.3.3 Maillard Browning ............................................................. 353 9.4 Milk Salts........................................................................................ 354 9.5 Vitamins.......................................................................................... 357 9.6 Proteins ........................................................................................... 357 9.6.1 Enzymes ............................................................................. 357 9.6.2 Denaturation of Other Biologically-Active Proteins .......... 359 9.6.3 Denaturation of Whey Proteins .......................................... 359 9.6.4 Effect of Heat on Caseins ................................................... 362 9.7 Heat Stability of Milk..................................................................... 364 9.7.1 Effect of Processing Operations on Heat Stability ............. 367 9.8 Effect of Heat Treatment on Rennet Coagulation of Milk and Related Properties....................................................... 369 9.9 Age Gelation of Sterilized Milk ..................................................... 370 9.10 Heat-Induced Changes in Flavour of Milk ..................................... 370 References................................................................................................. 373 Suggested Reading.................................................................................... 375

xviii Contents 10 Enzymology of Milk and Milk Products............................................... 377 10.1 Introduction .................................................................................... 377 10.2 Indigenous Enzymes of Bovine Milk ............................................. 378 10.2.1 Introduction..................................................................... 378 10.2.2 Proteinases (EC 3.4.-.-)................................................... 379 10.2.3 Lipases and Esterases (EC 3.1.1.-) ................................. 382 10.2.4 Phosphatases (EC 3.1.3 -)............................................... 384 10.2.5 Ribonuclease................................................................... 388 10.2.6 Lysozyme (EC 3.1.2.17) ................................................. 389 10.2.7 N-Acetyl-β-D-Glucosaminidase (EC 3.2.1.30) ............... 390 10.2.8 γ-Glutamyl Transpeptidase (Transferase) (EC 2.3.2.2) .. 390 10.2.9 Amylases (EC 3.2.1.-) .................................................... 391 10.2.10 Catalase (EC 1.11.1.6) .................................................... 391 10.2.11 Lactoperoxidase (EC 1.11.1.7) ....................................... 392 10.2.12 Xanthine Oxidoreductase (XOR) [EC, 1.13.22; 1.1.1.204] ................................................. 394 10.2.13 Sulphydryl Oxidase (EC 1.8.3.-) .................................... 396 10.2.14 Superoxide Dismutase (EC 1.15.1.1) ............................. 396 10.2.15 Other Enzymes................................................................ 397 10.3 Exogenous Enzymes in Dairy Technology..................................... 400 10.3.1 Introduction..................................................................... 400 10.3.2 Proteinases ...................................................................... 400 10.3.3 β-Galactosidase............................................................... 402 10.3.4 Lipases ............................................................................ 402 10.3.5 Lysozyme........................................................................ 403 10.3.6 Transglutaminase ............................................................ 404 10.3.7 Catalase (EC 1.1.1.6) ...................................................... 404 10.3.8 Glucose Oxidase (EC 1.1.3.4) ........................................ 405 10.3.9 Superoxide Dismutase (EC 1.15.1.1) ............................. 406 10.3.10 Glucose Isomerase (EC 5.3.1.5) ..................................... 406 10.3.11 Exogenous Enzymes in Food Analysis........................... 407 References and Suggested Reading .......................................................... 411 Indigenous Enzymes in Milk.......................................................... 411 Exogenous Enzymes in Dairy Technology and Analysis ............... 414 11 Biologically Active Compounds in Milk ............................................... 415 11.1 Introduction .................................................................................... 415 11.2 Bioactive Milk Lipids ..................................................................... 417 11.2.1 Medium Chain Fatty Acids ............................................. 417 11.2.2 Conjugated Linoleic Acid ............................................... 417 11.2.3 Polar Milk Lipids............................................................ 418 11.2.4 Fatty Acids with Significant Bioactivity......................... 418 11.2.5 Gangliosides ................................................................... 419 11.2.6 Milk Fat Globule Membrane .......................................... 419 11.2.7 Phospholipids.................................................................. 420

Contents xix 11.3 Bioactive Milk Carbohydrates ..................................................... 420 11.4 11.3.1 Lactose.......................................................................... 421 11.5 11.3.2 Oligosaccharides........................................................... 421 11.3.3 Bifidus Factors .............................................................. 422 11.6 11.3.4 Fucose ........................................................................... 422 Vitamins ....................................................................................... 423 11.7 Bioactive Milk Proteins................................................................ 424 11.8 11.5.1 Caseins.......................................................................... 424 11.9 11.5.2 Whey Proteins............................................................... 425 11.10 11.5.3 Vitamin-Binding Proteins ............................................. 428 11.5.4 Hormone-Binding Proteins........................................... 430 11.11 11.5.5 Metal-Binding Proteins................................................. 430 Minor Biologically-Active Proteins in Milk ................................ 431 11.12 11.6.1 Heparin Affinity Regulatory Peptide ............................ 432 11.6.2 Colostrinin .................................................................... 432 11.6.3 β2-Microglobulin........................................................... 433 11.6.4 Osteopontin................................................................... 433 11.6.5 Proteose Peptone-3 ....................................................... 433 11.6.6 Angiogenins.................................................................. 433 11.6.7 Kininogens.................................................................... 434 11.6.8 Glycoproteins................................................................ 434 Indigenous Milk Enzymes............................................................ 435 11.7.1 Lysozyme...................................................................... 435 11.7.2 Lactoperoxidase............................................................ 436 Bioactive Milk Peptides ............................................................... 436 11.8.1 Production of Bioactive Peptides.................................. 437 11.8.2 Physiological Functionality of Bioactive Peptides ....... 438 Free Amino Acids......................................................................... 457 Hormones, Growth Factors and Cytokines .................................. 458 11.10.1 Gonadal Hormones ....................................................... 461 11.10.2 Adrenal Hormones........................................................ 461 11.10.3 Brain-Gut Hormones .................................................... 461 11.10.4 Growth Factors.............................................................. 463 11.10.5 Cytokines ...................................................................... 466 11.10.6 Adipokins...................................................................... 467 Minor Bioactive Compounds ....................................................... 469 11.11.1 Polyamines.................................................................... 469 11.11.2 Amyloid A .................................................................... 470 11.11.3 Nucleotides ................................................................... 471 11.11.4 Calmodulin-Inhibiting Peptide ..................................... 471 11.11.5 Cluster of Differentiation 14 (CD14)............................ 471 11.11.6 Cysteine Protease Inhibitors ......................................... 472 11.11.7 Antioxidants and Prooxidants....................................... 472 Effect of Processing Conditions on Bioactive Components in Milk..................................................................... 472

xx Contents 11.13 Commercial Production and Uses of Bioactive 474 Compounds from Milk................................................................. 476 477 11.14 Bioactive Components in Other Milks......................................... 478 11.15 Conclusion.................................................................................... 497 References ................................................................................................. Suggested Reading.................................................................................... 12 Chemistry and Biochemistry of Cheese................................................ 499 12.1 Introduction .................................................................................... 499 12.2 Rennet-Coagulated Cheeses ........................................................... 501 12.2.1 Preparation and Treatment of Cheese Milk ...................... 501 12.2.2 Conversion of Milk to Cheese Curd ................................. 502 12.2.3 Acidification ..................................................................... 518 12.2.4 Moulding and Shaping...................................................... 521 12.2.5 Salting ............................................................................... 521 12.2.6 Manufacturing Protocols for Some Cheese Varieties ....... 522 12.2.7 Cheese Ripening ............................................................... 523 12.2.8 Cheese Flavour.................................................................. 533 12.2.9 Accelerated Ripening of Cheese....................................... 534 12.3 Acid-Coagulated Cheeses............................................................... 537 12.4 Processed Cheese Products............................................................. 537 12.4.1 Processing Protocol .......................................................... 541 12.5 Cheese Analogues........................................................................... 543 References................................................................................................. 544 Suggested Reading.................................................................................... 545 13 Chemistry and Biochemistry of Fermented Milk Products................ 547 13.1 Introduction .................................................................................... 547 13.1.1 Classification of Fermented Milks.................................... 548 13.1.2 Therapeutic Properties of Fermented Milks ..................... 550 13.2 Starter Microorganisms .................................................................. 553 13.3 Buttermilk....................................................................................... 554 13.4 Yoghurt ........................................................................................... 555 13.4.1 Concentrated Fermented Milk Products ........................... 555 13.4.2 Novel Yoghurt Products .................................................... 557 13.4.3 Rheology of Yoghurt......................................................... 557 13.4.4 Exocellular Polysaccharides ............................................. 557 13.5 Kefir ................................................................................................ 558 13.6 Koumiss .......................................................................................... 561 13.6.1 Technological Developments in Koumiss Manufacture ... 564 13.6.2 Koumiss-Like Products from Non-equine Milk ............... 565 13.7 Cultured/Sour Cream...................................................................... 566 References................................................................................................. 566 Suggested Reading.................................................................................... 567 Index................................................................................................................. 569

Chapter 1 Production and Utilization of Milk 1.1 Introduction Milk is a fluid secreted by the female of all mammalian species, of which there are more than 4,000, for the primary function of meeting the complete nutritional require- ments of the neonate of the species. In addition, milk serves several physiological functions for the neonate. Most of the non-nutritional functions of milk are served by proteins and peptides which include immunoglobulins, enzymes and enzyme inhibi- tors, binding or carrier proteins, growth factors and antibacterial agents. Because the nutritional and physiological requirements of each species are more or less unique, the composition of milk shows very marked inter-species differences. Of the more than 4,000 species of mammal, the milks of only ~180 have been analysed and of these, the data for only about 50 species are considered to be reliable (sufficient number of samples, representative sampling, adequate coverage of the lactation period). Not sur- prisingly, the milk of the principal dairying species, i.e., cow, goat, sheep and buffalo, and the human are among those that are well characterized. The gross composition of milks from selected species are summarized in Table 1.1; very extensive data on the composition of bovine and human milk are contained in Jensen (1995). 1.2 Composition and Variability of Milk In addition to the principal constituents listed in Table 1.1, milk contains several hundred minor constituents, many of which, e.g., vitamins, metal ions and flavour compounds, have a major impact on the nutritional, technological and sensoric properties of milk and dairy products. Many of these effects will be discussed in subsequent chapters. Milk is a very variable biological fluid. In addition to interspecies differences (Table 1.1), the milk of any particular species varies with the individuality of the © Springer International Publishing Switzerland 2015 1 P.F. Fox et al., Dairy Chemistry and Biochemistry, DOI 10.1007/978-3-319-14892-2_1

2 1 Production and Utilization of Milk Table 1.1 Composition, %, of milks of some species Species Total solids Fat Protein Lactose Ash Gross Days Human 12.2 3.8 1.0 7.0 0.2 energy to double Cow 12.7 3.7 3.4 4.8 0.7 (kJ/kg) birth weight Goat 12.3 4.5 2.9 4.1 0.8 Sheep 19.3 7.4 4.5 4.8 1.0 2,763 120–180 Pig 18.8 6.8 4.8 5.5 0.9 2,763 30–47 Horse 11.2 1.9 2.5 6.2 0.5 2,719 12–19 Donkey 11.7 1.4 2.0 7.4 0.5 4,309 10–15 Reindeer 33.1 16.9 11.5 2.8 1.5 3,917 9 Domestic rabbit 32.8 18.3 11.9 2.1 1.8 1,883 40–60 Bison 14.6 3.5 4.5 5.1 0.8 1,966 30–50 Indian elephant 31.9 11.6 4.9 4.7 0.7 6,900 22–25 Polar bear 47.6 33.1 10.9 0.3 1.4 9,581 4–6 Grey seal 67.7 53.1 11.2 0.7 0.8 – 100–115 3,975 100–260 16,900 2–4 20,836 5 animal, the breed (in the case of commercial dairying species), health (mastitis and other diseases), nutritional status, stage of lactation, age, interval between milkings, etc. The protein content of milk varies considerably between species and reflects the growth rate of the young. Bernhart (1961) found a linear correlation between the % calories derived from protein and the logarithm of the days to double birth weight for 12 mammalian species. For humans, one of the slowest growing and slowest maturing species, it takes 120–180 days to double birth weigh and only 7 % of calo- ries come from protein. In contrast, carnivores can double their birth weigh in as little as 7 days and obtain >30 % of their energy from protein. Equid species take between 30 and 60 days to double their birth weight and, like humans, have an exceptionally low level of protein in their milk (Table 1.1). The high calorific value of some species e.g., polar bear and grey seal is due mainly to the lipid content. In a bulked factory milk supply, variability due to many of these factors is evened out but some variability persists and may be quite large in situations where milk production is seasonal. Not only do the concentrations of the principal and minor constituents vary with the above factors, the actual chemistry of some of the con- stituents also varies, e.g., the fatty acid profile is strongly influenced by diet. Some of the variability in the composition and constituents of milk can be adjusted or counteracted by processing technology, e.g., standardization of fat content, but some differences may persist. The variability of milk and the consequent challenges will become apparent in subsequent chapters. From a physicochemical viewpoint, milk is a very complex fluid. The constituents of milk occur in three phases. Quantitatively, most of the mass of milk is a true solution of lactose, organic and inorganic salts, vitamins and other small molecules in water. In this aqueous solution are dispersed proteins, some at the molecular level (whey proteins), others as large colloidal aggregates, ranging in diameter from 50 to 600 nm (the caseins), and lipids which exist in an emulsified state, as globules ranging in diameter

1.3 Classification of Mammals 3 from 0.1 to 20 µm. Thus, colloidal chemistry is very important in the study of milk, e.g., surface chemistry, light scattering and rheological properties and phase stability. Milk is a dynamic system owing to: the instability of many of its structures, e.g., the milk fat globule membrane; changes in the conformation and solubility of many constituents with temperature and pH, especially of the inorganic salts but also of proteins; the presence of various enzymes which can modify constituents through lipolysis, proteolysis or oxidation/reduction; the growth of microorganisms, which can cause major changes either directly through their growth, e.g., changes in pH or redox potential (Eh) or through enzymes they excrete; and the interchange of gases with the atmosphere, e.g., CO2. Milk was intended to be consumed directly from the mammary gland and to be expressed from the gland at frequent intervals. However, in dairying operations, milk is stored for various periods, ranging from a few hours to several days, during which it is cooled (and perhaps heated) and agitated. These treatments will cause at least some physical changes and permit some enzymatic and microbiological changes which may alter the properties of milk. Again, it may be possible to counteract some of these changes. 1.3 Classification of Mammals The essential characteristic distinguishing mammals from other animal species is the ability of the female of the species to produce milk in specialized organs (mam- mary glands) for the nutrition of its newborn. The class Mammalia is divided into three sub-classes: 1. Prototheria This sub-class contains only one order, Monotremes, the species of which are egg-laying mammals, e.g., duck-billed platypus and echidna, and are indigenous only to Australasia. They possess many (perhaps 200) mammary glands grouped in two areas of the abdomen; the glands do not terminate in a teat and the secre- tion (milk) is licked by the young from the surface of the gland. 2. Marsupials The young of marsupials are born live (viviparous) after a short gestation and are “premature” at birth to a greater or lesser degree, depending on the species. After birth, the young are transferred to a pouch where they reach maturity, e.g., kan- garoo and wallaby. In marsupials, the mammary glands, which vary in number, are located within the pouch and terminate in a teat. The mother may nurse two off-spring, differing widely in age, simultaneously from different mammary glands that secrete milk of very different composition, designed to meet the different specific require- ments of each off-spring. 3. Eutherians About 95 % of all mammals belong to this sub-class. The developing embryo in utero receives nourishment via the placental blood supply (they are referred to as pla- cental mammals) and is born at a high, but variable, species-related state of maturity. All eutherians secrete milk, which, depending on the species, is more or less essential

4 1 Production and Utilization of Milk 102 Friesian Cow 101 100 Guernsey Cow 10–1 Horse 10–2 Buffalo Beef Cow Milk Yield (kg day–1) Goat Pig Sheep Rabbit Dog Man Rabbit Sheep Dog Baboon Fox Rat Guinea-Pig Hamster Echidna Mouse Tree Shrew 10–3 10–1 100 101 102 103 10–2 Body Weight (kg) Fig. 1.1 Relation between daily milk yield and maternal body weight for some species (modified from Linzell 1972) for the development of the young; the young of some species are born sufficiently mature to survive and develop without milk. The number and location of mammary glands varies with species from 2, e.g., human, goat and sheep, to 14–16 for the pig. Each gland is anatomically and physi- ologically separate and is emptied via a teat. The wide interspecies variation in the composition (Table 1.1) and the chemistry of the constituents of milk, as discussed elsewhere, renders milk species-specific, i.e., designed to meet the requirements of the young of that species. There is a surprisingly good relationship between milk yield and maternal body weight (Fig. 1.1); species bred for commercial milk production, e.g., dairy cow and goat, fall above the line. 1.4 Structure and Development of Mammary Tissue The mammary glands of all species have the same basic structure and all are located external to the body cavity (which greatly facilitates research on milk biosynthesis). Milk constituents are synthesized in specialized epithelial cells (secretory cells or mammocytes, Fig. 1.2d) from molecules absorbed from the blood. The secretory cells are grouped as a single layer around a central space, the lumen, to form more or less spherical or pear-shaped bodies, known as alveoli (Fig. 1.2c). The milk is secreted from these cells into the lumen of the alveoli. When the lumen is full, the myoepithe- lial cells surrounding each alveolus contract under the influence of oxytocin and the

1.4 Structure and Development of Mammary Tissue 5 Milk-producing tissue of a cow, shown at progressively larger scale ab BLOOD VESSEL ALVEOLUS SECRETORY TISSUE DUCTS CAPILLARIES DUCT CISTERN CONNECTIVE TISSUE c ARTERIAL BLOOD MILK-FAT DROPLETS LACTATING d CELL MILK PROTIEN CAPILLARIES ????? MYOEPITHELIAL GOLGI CELL APPARATUS LUMEN LIPID DROPLET MITOCHONDRION NUCLEUS ENDOPLASMIC RETICULUM DUCT Fig. 1.2 (a) A longitudinal section of one of the four quarters of a mammary gland; (b) arrange- ment of the alveoli and the duct system that drains them; (c) single alveolus consisting of an ellipti- cal arrangement of lactating cells surrounding the lumen, which is linked to the duct system of the mammary gland; (d) a lactating cell; part of the cell membrane becomes the membrane covering fat droplets; dark circular bodies in the vacuoles of Golgi apparatus are protein particles, which are discharged into the lumen (From Patton 1969)

6 1 Production and Utilization of Milk milk is drained via a system of arborizing ducts towards sinuses or cisterns (Fig. 1.2a) which are the main collection points between suckling or milking. The cisterns lead to the outside via the teat canal. Groups of alveoli, which are drained by a common duct, constitute a lobule; neighbouring lobules are separated by connective tissue (Fig. 1.2b). The secretory elements are termed the “lobule-alveolar system” to distin- guish them from the duct system. The whole gland is shown in Fig. 1.2a. Milk constituents are synthesized from components obtained from the blood; consequently, the mammary gland has a plentiful blood supply and an elaborate nervous system to regulate excretion. The substrates for milk synthesis enter the secretory cell across the basal mem- brane (outside), are utilized, converted and interchanged as they pass inwards through the cell and the finished milk constituents are excreted into the lumen across the apical membrane. Myoepithelial cells (spindle shaped) form a “basket” around each alveo- lus (Fig. 1.2c) and are capable of contracting on receiving an electrical, hormonally- mediated, stimulus, thereby causing ejection of milk from the lumen into the ducts. Development of mammary tissue commences before birth, but at birth the gland is still rudimentary. It remains rudimentary until puberty when very significant growth occurs in some species; in all species the mammary gland is fully developed at puberty. In most species, the most rapid phase of mammary gland development occurs at preg- nancy and continues through pregnancy and parturition, to reach a maximum at peak milk production. The data in Fig. 1.3 show the development pattern of the mammary gland in the rat, the species that has been studied most thoroughly in this regard. 40 Total mammary DNA, (mg) 30 Weaning 20 Parturition 10 Conception Puberty Birth 0 200 0 100 Days Fig. 1.3 Time-course of mammary development in rats (from Tucker 1969)

1.5 Ultrastructure of the Secretory Cell 7 ATROPHIC GLAND Fig. 1.4 The hormonal control of mammary Oest + GH + C development in rats. Oest oestrogen, Prog DUCT GROWTH progesterone, GH growth Oest + Prog + PL + hormone, PL prolactin, GH + C C corticosteroids LOBULO-ALVEOLAR GROWTH PL + C MILK SECRETION Mammary development is under the regulation of a complex set of hormones. Studies involving endocrinectomy (removal of different endocrine organs) show that the principal hormones are oestrogen, progesterone, growth hormone, prolactin and corticosteroids (Fig. 1.4). The anatomy, growth, development, involution and the gene network controlling these are described in a series of articles in Encyclopedia of Dairy Sciences, volume 3, pp 328–351 (Fuquay et al. 2011) 1.5 Ultrastructure of the Secretory Cell The structure of the secretory cell is essentially similar to that of other eukaryotic cells. In their normal state, the cells are roughly cubical, ~10 µm in cross section. It is estimated that there are ~5 × 1012 mammary cells in the udder of the lactating cow. A diagrammatic representation of the cell is shown in Fig. 1.2d. It contains a large nucleus towards the base of the cell and is surrounded by a cell membrane, the plas- malemma. The cytoplasm contains the usual range of organelles: Mitochondria: principally involved in energy metabolism [tricarboxylic acid (Kreb’s) cycle]. Endoplasmic reticulum: located towards the base of the cell and to which are attached ribosomes, giving it a rough appearance (hence the term, rough endoplasmic retic- ulum, RER). Many of the biosynthetic reactions of the cell occur in the RER.

8 1 Production and Utilization of Milk Golgi apparatus: a smooth membrane system located toward the apical region of the cell, where much of the assembly and “packaging” of synthesised material for excretion occur. Lysozyomes: capsules of enzymes (mostly hydrolytic) distributed fairly uniformly throughout the cytoplasm. Fat droplets and vesicles of material for excretion are usually apparent toward the apical region of the cell. The apical membrane possesses microvilli which serve to greatly increase its surface area. 1.6 Techniques Used to Study Milk Synthesis 1.6.1 Arterio-Venous Concentration Differences The artery and vein system supplying the mammary gland (Fig. 1.5) are readily accessible and may be easily cannulated to obtain blood samples for analysis. Differences in composition between arterial and venous blood give a measure of the c a 12 pv pa j h 3 4 A s V Fig. 1.5 The blood vessel and nerve supply in the mammary glands of a cow. Circulatory system (arteries, white; veins, stippled): h heart, a abdominal aorta, pa external pudic artery, pv external pudic vein, s subcutaneous abdominal vein, c carotid artery, j jugular vein. Nerves: 1 first lumbar nerve, 2 second lumbar nerve, 3 external spermatic nerve, 4 perineal nerve. A and V show blood sampling points for arteriovenous (AV) difference determinations (Mepham 1987)

1.6 Techniques Used to Study Milk Synthesis 9 constituents used in milk synthesis. The total amount of a constituent used may be determined if the blood flow rate is known, which may be easily done by infusing a known volume of cold saline solution into a vein and measuring the temperature of blood a little further down-stream. The extent to which the blood temperature is reduced is inversely proportional to blood flow rate. 1.6.2 Isotope Studies Injection of radioactively labelled substrates, e.g., glucose, into the blood stream permits assessment of the milk constituents into which that substrate is incorporated. It may also be possible to study the intermediates through which biosynthesis proceeds. 1.6.3 Perfusion of Isolated Gland In many species, the entire gland is located such that it may be readily excised intact and undamaged. An artificial blood supply may be connected to cannulated veins and arteries (Fig. 1.6); if desired, the blood supply may be passed through an artificial kidney. The entire mammary gland may be maintained active and secreting milk for several hours; substrates may be readily added to the blood sup- ply for study. 1.6.4 Tissue Slices The use of tissue slices is a standard technique in all aspects of metabolic biochem- istry. The tissue is cut into slices, sufficiently thin to allow adequate rates of diffu- sion in and out of the tissue. The slices are submerged in physiological saline to which substrates or other compounds may be added. Changes in the composition of the slices and/or incubation medium give some indication of metabolic activity but extensive damage may be caused to the cells on slicing; the system is so artificial that data obtained by the tissue slice technique may not pertain to the physiological situation. However, the technique is widely used at least for introductory, exploratory experiments. 1.6.5 Cell Homogenates Cell homogenates are an extension of the tissue slice technique, in which the tissue is homogenised. As the tissue is completely disorganized, only individual biosyn- thetic reactions may be studied in such systems; useful preliminary work may be done with homogenates.

10 1 Production and Utilization of Milk BP IA injections time-event recorder Teat cannula Heater Transducer V AV Thermistor- G thermometer Starling Water-bath bypass Infusion Gaddum pump flow recorder Venous samples Perfusion pump 95 5 O, CO, Arterial Substrate samples solution Oxygenator Fig. 1.6 Diagram of circuit for perfusion of an isolated mammary gland of a guinea-pig, G mam- mary gland, A artery, V veins (from Mepham 1987) 1.6.6 Tissue Culture Tissue cultures are useful for preliminary or specific work but are incomplete. In general, the specific constituents of milk are synthesized from small mole- cules absorbed from the blood. These precursors are absorbed across the basal membrane but very little is known about the mechanism by which they are trans- ported across the membrane. Since the membrane is rich in lipids and precursors are mostly polar with poor solubility in lipid, it is unlikely that the precursors enter the cell by simple diffusion. It is likely, in common with other tissues, that there are specialized carrier systems to transport small molecules across the membrane; such carriers are probably proteins. The mammary gland of the mature lactating female of many species is by far the most metabolically active organ of the body. For many small mammals, the energy input required for the milk secreted in a single day may exceed that required to develop a whole litter in utero. A cow at peak lactation yielding 45 kg milk/day secretes approximately 2 kg lactose and 1.5 kg each of fat and protein per day. This compares with the daily weight gain for a beef animal of 1–1.5 kg/day, 60–70 % of

1.8 Production and Utilization of Milk 11 which is water. In large measure, a high yielding mammal is subservient to the needs of its mammary gland to which it must supply not only the precursors for the synthesis of milk constituents but also an adequate level of high-energy-yielding substrates (ATP, UTP, etc.) required to drive the necessary synthetic reactions. In addition, minor constituents (vitamins and minerals) must be supplied. 1.7 Biosynthesis of Milk Constituents The constituents of milk can be grouped into four general classes according to their source: – Organ (mammary gland) and species-specific (e.g., most proteins and lipids) – Organ but not species-specific (lactose) – Species but not organ-specific (some proteins) – Neither organ- nor species-specific (water, salts, vitamins) The principal constituents (lactose, lipids and most proteins) of milk are synthe- sised in the mammary gland from constituents absorbed from blood. However, con- siderable modification of constituents occurs in the mammary gland; the constituents are absorbed from blood through the basal membrane, modified (if necessary) and synthesised into the finished molecule (lactose, triglycerides, proteins) within the mammocyte (mainly in the endoplasmic reticulum) and excreted from the mam- mocyte through the apical membrane into the lumen of the alveolus. The biosynthe- sis of the principal constituents of milk is described in a series of articles in the Encyclopedia of Dairy Sciences, volume 3, pp 352–380 (Fuquay et al. 2011) and in the appropriate chapter. 1.8 Production and Utilization of Milk Sheep and goats were domesticated early during the Agricultural Revolution, 8–10,000 years ago. Cattle were domesticated later but have become the principal dairying species, especially Bos taurus in the most intense dairying areas, dairy sheep and goats are widespread and very important in arid regions, especially around the Mediterranean. Buffalo are important in some regions, especially in India and Egypt. Dromedary camels are important dairy animals in North Africa and the Middle East. Mare’s milk is used extensively in central Asia and is receiving attention in Europe for special dietary purposes since its composition is closer to that of human milk than is bovine milk. Donkeys are also used for milk production on a small scale in several European countries, China and Ethiopia. Yak are particu- larly important in Western China, Mongolia and Tibet where they are used for trans- port, milk, meat and hides. Reindeer are of major significance in sub-Arctic regions. Approximately 85, 14, 2 and 1.5 % of world milk production is obtained from cattle,

12 1 Production and Utilization of Milk buffalo, goats and sheep, respectively, with very small proportions obtained from camels, yak, horse, donkey and reindeer. The animal species used for milk production are described in the Encyclopedia of Dairy Sciences, volume 1 pp 284–380 and the composition and properties of milk of selected species in volume 3, pp 478–631 (Fuquay et al. 2011). Some milk and dairy products are consumed in most, or all, regions of the World but they are major dietary items in Europe, North and South America, Australia, New Zealand and some Middle Eastern countries. Total milk production in 2013 was estimated to be 780 × 106 tonnes, of which 156, 100, 100 and 29 × 106 tonnes were produced in the European Union, Eastern Europe, North America and the Pacific region, respectively (FAO 2013). The European Union and some other coun- tries operate milk production quotas which are restricting growth in those areas; the quota system in the EU will cease in 2015 and milk production in the region is expected to increase. Data on the consumption of milk and dairy products in countries that are mem- bers of the International Dairy Federation (IDF) are summarized in Table 1.2. Milk and dairy products are quite important in several countries that are not included in this table since they are not members of the IDF. Because milk is perishable and its production was, traditionally, seasonal, milk surplus to immediate requirements was converted to more stable products, tradi- tional examples being butter or ghee, fermented milk and cheese; smaller amounts of dried milk products were produced traditionally by sun-drying. These traditional products are still very important and many new variants thereof have been intro- duced. In addition, several new products have been developed during the past 150 years, e.g., sweetened condensed milk, sterilized concentrated milk, a range of milk powders, UHT-sterilized milk, ice creams, infant foods and milk protein products. One of the important developments in dairy technology in recent years has been the fractionation of milk into its principal constituents, e.g., lactose, milk fat frac- tions and milk protein products (caseins, caseinates, milk protein concentrates, whey protein concentrates, whey protein isolates), mainly for use as functional pro- teins but recently some milk proteins, e.g., whey protein isolates, lactoferrin lacto- peroxidase and immunoglobulins are marketed as “nutraceuticals”, i.e., proteins for specific physiological and/or nutritional functions, As a raw material, milk has many attractive features: 1. Milk was designed for animal nutrition and hence contains the necessary nutri- ents in easily digestible forms (although the balance is designed for the young of a particular species) and free of toxins. No other single food, except the whole carcass of an animal, including the bones, contains the complete range of nutri- ents at adequate concentrations. 2. The principal constituents of milk, i.e., lipids, proteins and carbohydrates, can be readily fractionated and purified by relatively simple methods, for use as food ingredients. 3. Milk itself is readily converted into products with highly desirable organoleptic and physical characteristics and its constituents have many very desirable and some unique physicochemical (functional) properties.

1.8 Production and Utilization of Milk 13 Table 1.2 Consumption of liquid milk (L/caput/annum), cheese butter and fermented milks (kg/caput/annum) Country Milk Cheese Butter Fermented milk European Union Austria 75.2 19.2 5.0 21.8 Belgium 48.9 15.3 2.5 10.5 Croatia 71.2 9.6 1.0 16.9 Cyprus 97.9 18.1 1.9 12.4 Czech Republic 56.7 16.6 5.2 16.3 Denmark 87.2 16.1 1.8 48.2 Estonia 120.9 20.8 4.1 Finland 128.3 23.7 4.5 – France 52.6 26.2 7.4 38.6 Germany 53.3 24.3 6.2 29.9 Greece 47.6 23.4 0.7 30.5 Hungary 49.0 11.5 1.0 Ireland 135.6 6.7 2.4 – Italy 52.7 20.9 2.3 13.9 Latvia 91.9 16.0 2.8 Lithuania 29.5 16.3 2.8 – Luxemburg 36.8 24.4 6.1 8.8 Netherlands 47.5 19.4 3.3 – Poland 40.9 11.4 4.1 – Portugal 78.5 9.6 1.8 Slovakia 53.2 10.1 2.9 – Spain 80.6 9.3 0.6 45.0 Sweden 89.2 19.7 1.8 United Kingdom 102.9 11.2 3.4 7.8 Other European 26.6 Iceland 96.0 25.2 4.9 13.8 Norway 83.9 17.7 3.2 29.1 Switzerland 64.9 21.1 5.2 36.4 Russia 70.0 6.6 2.8 Ukraine 19.3 4.2 2.1 – Africa and Asia China 15.4 0.1 0.1 37.9 Egypt 23.7 9.4 0.7 25.5 India 40.0 2.4a 3.6 31.4 Iran 18.4 4.7 0.3 Israel 53.7 17.1 0.9 – Japan 30.6 2.1 0.6 11.7 Kazakhstan 25.6 2.5 1.1 Mongolia 8.9 0.3 0.6 1.9 South Africa 23.1 1.5 0.3 – – 47.3 23.3 – – – 1.8 (continued)

14 1 Production and Utilization of Milk Table 1.2 (continued) Country Milk Cheese Butter Fermented milk South Korea 34.9 2.0 0.2 9.3 Turkey 16.0 7.2 0.7 – Americas Argentina 41.1 11.2 1.4 12.8 Brazil 57.2 3.6 0.4 – Canada 77.0 12.1 2.8 8.2 Chile 22.3 8.1 1.2 – Colombia 59.4 0.9 0.1 – Mexico 34.8 3.1 0.3 5.3 Uruguay 67.1 6.0 1.6 – USA 74.0 15.2 2.5 – Oceania Australia 105.9 11.8 4.0 6.7 New Zealand 6.0 6.7 4.7 – Milk, cheese and butter data from IDF (2010, 2011) and Statistics Canada (2012) Fermented milk data from IDF (2009) aFrom Jayadevan (2013) 4. The modern dairy cow is a very efficient convertor of plant material; average national yields, e.g., in the USA and Israel, are about 8,000 kg per annum, with individual cows producing up to 20,000 kg per annum. In terms of kg protein that can be produced per hectare, milk production, especially by modern cows, is much more efficient than meat production (Fig. 1.7) but less efficient than some plants (e.g., cereals and soy beans). However, the functional and nutritional properties of milk proteins are superior to those of soy protein and since cattle, and especially sheep and goats, can thrive under farming conditions not suitable for growing cereals or soy beans, dairy animals need not be competitors with humans for use of land although high-yielding dairy cows are fed products that could be used for human foods. In any case, dairy products improve the “quality of life”, which is a desirable objective per se. 5. One of the limitations of milk as a raw material is its perishability—it is an excel- lent source of nutrients for microorganisms as well as for humans. However, this perishability is readily overcome by a well-organized, efficient dairy industry. Milk is probably the most adaptable and flexible of all food materials, as will be apparent from Table 1.3 which shows the principal families of milk-based foods— some of these families contain several hundred different products. Many of the processes to which milk is subjected cause major changes in the composition (Table 1.4), physical state, stability, nutritional and sensoric attributes of the product; some of these changes will be discussed in later chapters.

1.9 Trade in Milk Products 15 6 Days, 1000 5 4 3 2 1 0 Beef cattle Pigs Poultry Milk Cornflakes Oat meal Rye Flour Wheat flour (white) Rice (white) Maize meal Rice (brown) Wheat flour (whole) Beans (dry, edible) Peas (split) Soybeans Selected food products Fig. 1.7 Number of days of protein supply for a moderately active man produced per hectare yielding selected food products 1.9 Trade in Milk Products Milk and dairy products have been traded for thousands of years and are now major items of trade. There is considerable international trade in dairy products, principally, whole and skim milk powders, cheese, butter, whey protein powders and infant formulae. Data for the production, exports and imports (million tonnes milk equivalent) of milk for 2012 are provided by FAO (2013) and are summarized in Table 1.5. Traditionally, dairy products (cheese, fermented milks, butter) were produced on an artisanal level, as is still the case in underdeveloped regions and to some extent in highly developed dairying countries. Industrialization commenced during the nineteenth Century and dairy manufacturing is now a well organized industry. One of the features of the past few decades has been the amalgamation of smaller dairy companies both within countries, and, recently, internationally. The 20 largest dairy companies are listed in Table 1.6. An notable feature of these data is that the top 20

16 1 Production and Utilization of Milk Table 1.3 Diversity of dairy products Process Primary product Further products Cream Butter, butter oil, ghee, anhydrous milk fat; creams Centrifugal of various fat content: coffee creams, whipping Separation creams, dessert creams; cream cheeses Powders, casein, cheese, protein concentrates Skim milk and infant formulae HTST or super-pasteurization, UHT-sterilized Thermal processing or in-container sterilized Evaporated or sweetened condensed milk Concentration Cheese Thermal Whole milk powders; infant formulae; dietary evaporation or Rennet casein products Membrane Whey 1,000 varieties; further products, e.g., processed filtration cheese, cheese sauces, cheese dips Cheese analogues Concentration and Whey powders, demineralized whey powders, drying whey protein concentrates, whey protein isolates, individual whey proteins, whey protein Enzymatic hydrolyzates, nutraceuticals coagulation Lactose and lactose derivatives Fresh cheeses and cheese-based products Acid coagulation Cheese Functional applications, e.g., coffee creamers, meat Acid casein extenders; nutritional applications, cream liquers Whey powders, demineralized whey powders, Whey whey protein concentrates, whey protein isolates, individual whey proteins, whey protein Fermentation hydrolyzates, nutraceuticals Freezing Various fermented milk products, e.g., yoghurt, Miscellaneous buttermilk, acidophilus milk, bioyoghurt Ice cream (numerous types and formulations), frozen yoghurt Chocolate products companies process only 24.2 % of total milk production and the largest company only 3.0 %. Such developments have obvious advantages in terms of efficiency and standardization of product quality but pose the risk of over-standardization with the loss of variety. Greatest diversity occurs with cheeses and fortunately in this case, diversity is being preserved and even extended.

1.9 Trade in Milk Products 17 Table 1.4 Approximate composition (%) of some dairy products Product Moisture Protein Fat Sugarsa Ash 63.5 2.2 30.9 3.0 0.5 Light whipping cream 15.9 0.85 81.1 0.06 2.1 Butter 0.2 0.3 99.5 0.0 0.0 Anhydrous butter oil 60.8 3.6 10.8 23.8 1.0 Ice creamb 74.0 6.8 7.6 10.0 1.5 Evaporated whole milk 27.1 7.9 8.7 54.4 1.8 Sweetened condensed milk 26.3 26.7 38.4 6.1 Whole milk powder 2.5 36.2 0.8 52.0 7.9 Skim milk powder 3.2 8.3 Whey powderc 3.2 12.9 1.1 74.5 3.8 Casein powder 7.0 88.5 0.2 0.0 1.4 Cottage cheese, creamed 79.0 12.5 4.5 2.7 – Quarg 72.0 18.0 8.0 3.0 3.7 Camembert cheese 51.8 19.8 24.3 0.5 5.1 Blue cheese 42.4 21.4 28.7 2.3 3.9 Cheddar cheese 36.7 24.9 33.1 1.3 – Emmental cheese 36.0 28.9 30.0 – 6.0 Parmesan cheese 29.2 35.7 24.8 3.2 2.6 Mozzarella cheese 54.1 19.4 31.2 2.2 5.8 Processed cheesed 39.2 22.1 31.2 1.6 – Acid whey 93.9 0.6 0.2 4.2 aTotal carbohydrate bHardened vanilla, 19 % fat cCheddar (sweet) whey dAmerican pasteurized processed cheese Table 1.5 Production, exports and imports (millions of tonnes of milk equivalents) for 2012 (FAO 2013) Country Production Imports Exports 27.8 5.7 Asia 90.2 8.8 1.2 0.5 Africa 45.8 4.4 3.8 3.8 5.4 Central America 16.5 1.7 5.9 16.2 South America 68.2 0.85 20.7 53.4 53.4 North America 99.3 Europea 216.3 Oceania 29.3 World 765.6 aTrade between EU countries is not included

18 1 Production and Utilization of Milk Table 1.6 Ranking of global dairy processors, 2011 (from Jesse 2013) Rank Company name Headquarter Turnover Milk intake Mkt share (% country ($US Bil.) (Mil. MT) worldmilk prod.) 1 Nestlé Switzerland 19.1 14.9 2.1 16.9 15.0 2.1 2 Parmalat France 16.4 21.6 3.0 15.6 8.2 1.1 3 Fonterra Coop. New Zealand 13.4 10.1 1.4 13.0 17.1 2.4 4 Danone France 13.0 12.1 1.7 12.0 12.0 1.7 5 Friesland-Campina Netherlands 1.1 7.5 7.8 0.9 6 DFAa USA 7.0 6.3 0.6 6.5 4.4 1.0 7 Dean Foods USA 6.4 6.9 0.6 5.8 4.1 0.6 8 Arla Group Denmark 5.8 4.0 0.6 5.7 4.1 0.5 9 Kraft Foods USA 5.5 3.6 0.8 4.3 5.9 0.8 10 Saputo Canada 3.9 6.0 0.6 3.0 4.6 0.6 11 Müller Germany 2.5 4.0 12 DMKb Germany 13 Mengniu Dairy Co. China 14 Yili China 15 Groupe Sodiaal France 16 Bongrain SA France 17 Land O’ Lakes, Inc. USA 18 Glanbia Group Ireland 19 California Dairies USA 20 Amul (Coop.) India aDairy Farmers of America bDeutsches Milchkontor GmbH References Bernhart, F. W. (1961). Correlations between growth-rate of the suckling of various species and the percentage of total calories from protein in the milk. Nature, 191, 358–360. FAO. (2013). Food outlook. Rome: FAO. Fuquay, J., Fox, P. F., & McSweeney, P. L. H. (Eds.) (2011). Encyclopedia of dairy sciences (Vol. 3, pp. 328–351, 352–380, 478–631). Oxford: Elsevier Academic Press. IDF. (2009). The world dairy situation. Bulletin 438/2009. International Dairy Federation, Brussels. IDF. (2010). The world dairy situation. Bulletin 446/2010. International Dairy Federation, Brussels. IDF. (2011). The world dairy situation. Bulletin 451/2011. International Dairy Federation, Brussels. Jayadevan, G. R. (2013). A strategic analysis of cheese and cheese products market in India. Indian Journal of Research, 2, 247–250. http://theglobaljournals.com/paripex/file.php?val=March_20 13_1363940771_5f9c2_83.pdf. Jensen, R. G. (Ed.). (1995a). Handbook of milk composition. San Diego, CA: Academic. Jesse, E. V. (2013, February). International dairy notes. The Babcock Institute Newsletter. College of Agricultural and Life Sciences, University of Wisconsin, Madison, USA. Linzell, J. L. (1972). Milk yield, energy loss, and mammary gland weight in different species. Dairy Science Abstracts, 34, 351–360. Mepham, T. B. (1987a). Physiology of lactation. Milton Keynes, UK: Open University Press. Patton, S. (1969). Milk. Scientific American, 221, 58–68. Statistics Canada. (2012). Dairy statistics. Ottawa: Government of Canada. Tucker, H. A. (1969). Factors affecting mammary gland cell numbers. Journal of Dairy Science, 52, 720–729.

Suggested Reading 19 Suggested Reading Cowie, A. T., & Tindal, J. S. (1972). The physiology of lactation. London, UK: Edward Arnold. Fuquay, J., Fox, P. F., & McSweeney, P. L. H. (Eds.). (2011b). Encyclopedia of dairy sciences (2nd ed., Vol. 1–4). Oxford, UK: Academic. Jensen, R. G. (Ed.). (1995b). Handbook of milk composition. San Diego, CA: Academic. Larson, B. L. & Smith, V. R. (1974–1979). Lactation: A comprehensive treatise (Vols. 1–4). New York: Academic Press. Mepham, T. B. (1975). The secretion of milk (Studies in biology series, Vol. 60). London, UK: Edward Arnold. Mepham, T. B. (Ed.). (1983). Biochemistry of lactation. Amsterdam: Elsevier. Mepham, T. B. (1987b). Physiology of lactation. Milton Keynes, UK: Open University Press. Park, Y. W., & Haenlein, G. F. W. (Eds.). (2013). Milk and dairy products in human nutrition. Chichester, UK: Wiley Blackwell. Singh, H., Boland, M., & Thompson, A. (Eds.). (2014). Milk proteins: From expression to food (2nd ed.). Amsterdam: Academic.

Chapter 2 Lactose 2.1  Introduction Lactose is the principal carbohydrate in the milk of most mammals, exceptions are the California sea lion and the hooded seal, which are the only significant sources. Milk contains only trace amounts of other sugars, including glucose (50 mg/l) and fructose and glucosamine, galactosamine and N-acetyl neuraminic acid as compo- nents of glycoproteins and glycolipids. The milk of all species that have been studied contain oligosaccharides which are major constituents of the milk of some species, including human. This chapter will concentrate on the chemistry and properties of lactose with a short section on oligosaccharides. The concentration of lactose in milk varies widely between species (Table 2.1). The lactose content of cows' milk varies with the breed of cow, individual animals, udder infection (mastitis) and stage of lactation. The concentration of lactose decreases progressively and significantly during lactation (Fig. 2.1); this behaviour contrasts with the trends for lipids and proteins, which, after decreasing during early lactation, increase strongly during the second half of lactation. The concentration of lactose in milk is inversely related to the concentrations of lipids and proteins (Fig 2.2) (Jenness and Sloan 1970; Jenness and Holt 1987). The principal function of lactose and lipids is as sources of energy; since lipids are ~2.2 times more energy-d­ ense than lactose, when a highly caloric milk is required, e.g., by animals in a cold environment (marine mammals and polar bears), this is achieved by increasing the fat content of the milk. The inverse relationship between the concentrations of lactose and lipids and protein reflects the fact that the synthesis of lactose draws water into the Golgi vesicles, thereby diluting the concentrations of proteins and lipids (Jenness and Holt 1987). Mastitis causes an increased level of NaCl in milk and depresses the secretion of lactose. Lactose, along with sodium, potassium and chloride ions, plays a major role in maintaining the osmotic pressure in the mammary system. Thus, any increase or decrease in lactose content (a secreted constituent, i.e., formed within the mammary gland, which is isotonic with blood) is compensated for by an increase or decrease © Springer International Publishing Switzerland 2015 21 P.F. Fox et al., Dairy Chemistry and Biochemistry, DOI 10.1007/978-3-319-14892-2_2

22 2 Lactose Table 2.1  Concentration (%) of lactose in the milk of selected species Species Lactose Species Lactose Species Lactose California sea lion 0.0 Mouse (house) 3.0 Cat (domestic) 4.8 Hooded seal 0.0 Guinea pig 3.0 Pig 5.5 Black bear 0.4 Dog (domestic) 3.1 Horse 6.2 Dolphin 0.6 Sika deer 3.4 Chimpanzee 7.0 Echidna 0.9 Goat 4.1 Rhesus monkey 7.0 Blue whale 1.3 Elephant (Indian) 4.7 Man 7.0 Rabbit 2.1 Cow 4.8 Donkey 7.4 Red deer 2.6 Sheep 4.8 Zebra 7.4 Grey seal 2.6 Water buffalo 4.8 Green monkey 10.2 Rat (Norwegian) 2.6 6 5 Percent 4 3 0 10 20 30 40 50 60 Week Fig. 2.1  Changes in the concentrations of fat (closed triangle), protein (empty square) and lactose (open circle) in milk during lactation in the soluble salt constituents (excreted) (Fig. 2.3). This osmotic relationship partly explains why certain milks with a high lactose content have a low ash content and vice versa (Table 2.2). Similarly, there is an inverse relationship between the concentrations of l­actose and chloride, which is the basis of Koestler’s chloride-lactose test for abnormal milk: Koestler Number = %Chloride ´100 %Lactose A Koestler Number <2 indicates normal milk while a value >3 is considered abnormal.

2.1 Introduction 23 aLactose, % 7Lactose, % 6 5 4 3 2 1 0 -1 -2 0 10 20 30 40 50 60 Fat, % b8 7 6 5 4 3 2 1 0 0123456789 Casein, % Fig. 2.2  Correlation between lactose and fat (a) and casein (b) in the milk of 23 species (based on the data of Jenness and Sloan 1970) Lactose plays an important role in milk and milk products: 1. It is an essential constituent in the production of fermented dairy products. 2. It contributes to the nutritive value of milk and its products; however, many ­non-E­ uropeans have limited or zero ability to digest lactose in adulthood, ­leading  to lactose intolerance. 3 . It affects the texture of certain concentrated and frozen products. 4 . It is involved in heat-induced changes in the colour and flavour of highly heated milk products. 5. Its changes in state (amorphous vs. crystalline) have major implications for the production and stability of many dehydrated milk products.

24 2 Lactose 140 130 Lactose, mM 120 110 100 90 100 110 120 130 90 Salt Osmolarity, mM Fig. 2.3  Relationship between the concentration of lactose (mM) and osmolarity (mM) due to salts (redrawn from the data of Holt 1985) Table 2.2  Average concentration (%) of lactose and ash in the milk of some mammals Species Water Lactose Ash Human 87.4 6.9 0.21 Cow 87.2 4.9 0.70 Goat 87.0 4.2 0.86 Camel 87.6 3.26 0.70 Mare 89.0 6.14 0.51 Reindeer 63.3 2.5 1.40 2.2  Chemical and Physical Properties of Lactose 2.2.1  Structure of Lactose Lactose is a disaccharide consisting of galactose and glucose, linked by a β1-4 glycosidic bond (Fig. 2.4). Its systematic name is 0-β-d-galactopyranosyl-(1-4)-α- d-g­ lucopyranose (α-lactose) or 0-β-d-galactopyranosyl-(1-4)-β-d-glucopyranose (β-lactose). The hemiacetal group of the glucose moiety is potentially free (i.e., lactose is a reducing sugar) and may exist as an α- or β-anomer. In the struc- tural formula of the α-form, the hydroxyl group on the C1 of glucose is cis to the hydroxyl group at C2 (oriented downward).

2.2  Chemical and Physical Properties of Lactose 25 b a H H OH a HO H 1C 1C 1C O HO 2 C H H 2C OH HO 3C H O H 3C OH O HO 4C H 4 CH H 5C H 5C 6 CH2OH 6 CH2OH b (1 4) 6 CH2OH Anomeric carbon O O OH 6 CH2OH H5 O 1 O H H 1 b HO H5 H H 4b 2H 4 H 1a 2H OH 2 OH OH 3 H3 OH OH H OH H Lactose O O O O-b-D-Galactopyranosyl-(1 4)-a-D-Glucopyranose : a-Lactose a Galactose b (1 O-b-D-Galactopyranosyl-(1 4)-b-D-Glucopyranose : b-Lactose OH O 4) Glucose O OOH b c OH 6CH2OH O 6 O 1H OH 4 HOH2C 5 a 1 H 5H H H H2 4 H 2 OH b HO 3 1O H 3 OH H HO OH H H Fig. 2.4  Structural formulae of α- and β-lactose (a) Open chain, (b) Fischer projection, (c) Haworth projection and (d) conformational formula

26 2 Lactose GLUCOSE hexokinase Glucose-6-phosphate ATP ADP Phosphoglucomutase P-P UDP-glucose UDP glucose Glucose-1-phosphate pyrophosphorylase UDP glucose-4-epimerase UTP ADP ATP UDP LACTOSE UDP-galactose galactosyltransferase Glucose a-lactalbumin Fig. 2.5  Pathway for lactose synthesis 2.2.2  Biosynthesis of Lactose Lactose is unique to mammary secretions. It is synthesized from glucose absorbed from blood. One molecule of glucose is isomerized to UDP-galactose via the 4-enzyme Leloir pathway (Fig. 2.5). UDP-Gal is then linked to another molecule of glucose in a reaction catalysed by the enzyme, lactose synthetase, a 2-component enzyme. Component A is a non-specific galactosyl transferase (EC 2.4.1.22) which transfers the galactose from UDP-gal to a number of accep- tors. In the presence of the B component, which is the whey protein, α-lactalbumin, the transferase becomes highly specific for glucose (its KM is decreased 1,000-fold), leading to the synthesis of lactose. Thus, α-lactalbumin is an enzyme modifier and its concentration in milk is directly related to the concentration of lactose (Fig. 2.6); the milk of some marine mammals contain neither α-lactalbumin nor lactose. The presumed significance of this control mechanism is to enable mammals to terminate the synthesis of lactose when necessary, i.e., to regulate and con- trol osmotic pressure when there is an influx of NaCl, e.g., during mastitis or in late lactation (lactose and NaCl are the major determinants of the osmotic pres- sure of milk, which is isotonic with blood, the osmotic pressure of which is essentially constant). The ability to control osmotic pressure is sufficiently important to justify an elaborate control mechanism and \"wastage\" of the enzyme modifier.

2.2  Chemical and Physical Properties of Lactose 27 Fig. 2.6  Correlation between lactose and α-lactalbumin concentrations in the milk of eight spe- cies (adapted from Ley and Jenness 1970) 2.2.3  Lactose Equilibrium in Solution The configuration around the C1 of glucose (i.e., the anomeric C) is not stable and can readily change (mutarotate) from the α- to the β-form and vice versa when the sugar is in solution as a consequence of the fact that the hemiacetal form is in equi- librium with the open chain aldehyde form which can be converted into either of the two isomeric forms (Fig. 2.4). When either isomer is dissolved in water, there is a gradual change from one form to the other until equilibrium is established, i.e., mutarotation occurs. These changes may be followed by measuring the change in optical rotation with time until, at equilibrium, the specific rotation is +55.4°. The composition of the mixture at equilibrium may be calculated as follows: Specific rotation: [α]D20 α-form +89.4° β-form +35.0° Equilibrium mixture +55.4° Let equilibrium mixture = 100 Let x% of the lactose be in the α-form Then (100 - x)% is the β-form At equilibrium:  89.4x + 35(100 − x) = 55.4 × 100  x = 37.5  100 − x = 62.5

28 2 Lactose Thus, the equilibrium mixture at 20 °C is composed of 62.7 % β and 37.3 % α-lactose. The equilibrium constant, β/α, is 1.68 at 20 °C. The proportion of lactose in the α-form increases as the temperature is increased and the equilibrium constant consequently decreases. The equilibrium constant is not influenced by pH, but the rate of mutarotation is dependent on both temperature and pH. The change from α- to β- is 51.1, 17.5 and 3.4 % complete at 25, 15 and 0 °C, respectively, in 1 h and is almost instantaneous at about 75 °C. The rate of mutarotation is slowest at pH 5.0, increasing rapidly at more acid or alkaline values; equilibrium is established in a few minutes at pH 9.0. 2.2.4  S ignificance of Mutarotation The α and β forms of lactose differ with respect to: Solubility Crystal shape and size Hydration of the crystalline form, which leads to hygroscopicity Specific rotation Sweetness Many of these characteristics are discussed in the following sections. 2.2.5  S olubility of Lactose The solubility characteristics of the α- and β-isomers are distinctly different. When α-lactose is added in excess to water at 20 °C, about 7 g per 100 g water dissolve immediately. Some α-lactose mutarotates to the β anomer to establish the equilib- rium ratio 62.7β:37.3α; therefore, the solution becomes unsaturated with respect to α and more α-lactose dissolves and some mutarotetes to β-lactose. These two pro- cesses (mutarotation and solubilization of α-lactose) continue until two criteria are met: ~7 g α-lactose are in solution and the β/α ratio is 1.6:1.0. Since the β/α ratio at equilibrium is about 1.6 at 20 °C, the final solubility is 7 g + (1.6 × 7) g = 18.2 g per 100 g water. When β-lactose is dissolved in water, the initial solubility is ~50 g per 100 g water at 20 °C. Some β-lactose mutarotates to α to establish a ratio of 1.6:1. At equilibrium, the solution would contain 30.8 g β and 19.2 g α/100 ml; therefore, the solution is supersaturated with α-lactose, some of which crystallizes, upsetting the equilibrium and leading to further mutarotation of β to α. These two events, i.e., crystallization of α-lactose and mutarotation of β, continue until the same two crite- ria are met, i.e., ~7 g of α-lactose in solution and a β/α ratio of 1.6:1. Again, the final solubility is ~18.2 g lactose per 100 g water. Since β-lactose is much more soluble than α and mutarotation is slow, it is possible to form more highly concentrated solutions by dissolving β- rather than α-lactose. In either case, the final solubility of lactose is the same (18.2 g/100 g of water).

2.2  Chemical and Physical Properties of Lactose 29 The solubility of lactose as a function of temperature is summarized in Fig. 2.7. The solubility of α-lactose is more temperature dependent than that of β-lactose and the solubility curves intersect at 93.5 °C. A solution at 60 °C contains approxi- mately 59 g lactose per 100 g water. Suppose that a 50 % solution of lactose (~30 g β- and 20 g α-) at 60 °C is cooled to 15 °C. At this temperature, the solution can contain only 7 g of α- or a total of 18.2 g of lactose per 100 g water at equilibrium. Therefore, lactose will crystallize very slowly out of solution as irregularly-sized crystals which may give rise to a sandy, gritty texture. 2.2.6  Crystallization of Lactose As discussed in Sect. 2.2.5, the solubility of lactose is temperature dependent and solu- tions are capable of being highly supersaturated before spontaneous crystallization occurs and even then, crystallization may be slow. In general, supersolubility at any temperature equals the saturation (solubility) value at a temperature 30 °C higher. The insolubility of lactose, coupled with its capacity to form supersaturated solutions, is of considerable practical importance in the manufacture of concentrated milk products. In the absence of nuclei and agitation, solutions of lactose are capable of being highly supersaturated before spontaneous crystallization occurs. Even in such solu- tions, crystallization occurs with difficulty. Solubility curves for lactose are shown in Fig. 2.8 and are divided into unsaturated, metastable and labile zones. Cooling a satu- rated solution or continued concentration beyond the saturation point, leads to super- saturation and produces a metastable area where crystallization does not occur readily. At higher levels of supersaturation, a labile area is observed where crystallization occurs readily. The pertinent points regarding supersaturation and crystallization are: 1 . Neither nucleation nor crystal growth occurs in the unsaturated region. 2. Growth of crystals can occur in both the metastable and labile areas. 3. Nucleation occurs in the metastable area only if seeds (centres for crystal growth) are added. 4. Spontaneous crystallization can occur in the labile area without the addition of seeding material. The rate of nucleation is slow at low levels of supersaturation and in highly supersaturated solutions owing to the high viscosity of the solution. The stability of a lactose “glass” (see Sect. 2.2.6.4) is due to the low probability of nuclei forming at very high concentrations. Once a sufficient number of nuclei have formed, crystal growth occurs at a rate influenced by: (a) Degree of supersaturation. ( b) Surface area available for deposition. (c) Viscosity. ( d) Agitation. (e) Temperature. (f) Mutarotation, which is slow at low temperatures.

30 2 Lactose Final solubility at equilibrium 160 Solubility, g anhydrous lactose / 100 g water 140 120 100 Initial solubility of β-lactose Usual range of 80 supersaturation 60 40 93.5°C 20 Initial solubility of α-lactose 0 0 20 40 60 80 100 Temperature, (°C) Fig. 2.7  Solubility of lactose in water (modified from Jenness and Patton 1959) 200 Solubility (g lactose/100 g water) 100 2.1 1 1.6 labile intermmeedtaiastteanobtlesaturated β α 40 20 10 5 0 20 40 60 80 100 Temperature, (°C) Fig. 2.8  Initial solubility of α-lactose and β-lactose, final solubility at equilibrium (line 1), and supersaturation by a factor 1.6 and 2.1 (α-lactose excluding water of crystallization) (modified from Walstra and Jenness 1984)